Conflicts of interest: the author has not declared any conflicts of interest.
Rapid genetic changes in natural insect populations
Article first published online: 4 JAN 2010
© 2010 The Author. Journal compilation © 2010 The Royal Entomological Society
Special Issue: Insect Evolution Below the Species Level
Volume 35, Issue Supplement s1, pages 155–164, January 2010
How to Cite
LOXDALE, H. D. (2010), Rapid genetic changes in natural insect populations. Ecological Entomology, 35: 155–164. doi: 10.1111/j.1365-2311.2009.01141.x
- Issue published online: 4 JAN 2010
- Article first published online: 4 JAN 2010
- Accepted 6 October 2009
- molecular markers;
- natural selection;
- sub-specific variation
1. The insects represent around 75% of the world's fauna and as such provide especially good examples of the evolutionary process in action, aided by their often rapid generation time and high rate of reproduction.
2. Here, I review some of the main mechanisms of mutational, ecological, and evolutionary change in insects. All those described, whether allo- para- or sympatric, involve changes in the genome or in behaviour that may ultimately isolate newly changed individuals from the parental population/s. They include: loss of sexuality by various means, including (potentially) mutation of the gene/s controlling sexuality; karyotypic changes, both in terms of the number of chromosomes, translocation, polyploidy, and hybridisation; host shifts as pre- and post-zygotic isolating mechanisms, and in asexuals such as aphids, ‘divergence hitchhiking’ around key quantitative trait loci (QTL), and in moths, selection acting at a few linkage groups; enzyme-based adaptive changes; sex and contact pheromone-based isolating mechanisms; phenotypic plastic changes; and epigenetic changes. These last such changes are brought about by stress inducers, and may be transgenerational in their effects.
3. Of the above mechanisms, probably chromosomal changes and host shifts represent the commonest mechanisms.
Insects represent a huge part of global biodiversity, something like 75% of the entire world's fauna (Grimaldi & Engel, 2005), and occupy most of the world's biomes, with the exception of the marine environment, although even here they have made inroads (Cheng, 1976). To date, something like a million species have been described, but total estimates put this much higher (cf. Tokeshi, 1998). Because of this diversity, including a wide range of structures, behaviour, and life cycles, often with different reproductive strategies, in turn sometimes involving asexual phases, insects are excellent model organisms for studies on ecology and evolution. This includes below the species level, in effect, the cutting edge of the population divergence process. In recent decades, the arrival of molecular markers, notably DNA, have facilitated and enhanced such studies (Loxdale & Lushai, 1998) and indeed, may be described as the finest level of resolution available for much ecological research.
In the present paper, I briefly outline the main mechanisms of insect population divergence discovered to date, elucidated using chromosomal, molecular genetic, and sometimes chemical markers from a series of case studies, including aphids, my own main research interest. I begin very briefly by discussing whether aphid clones are subject to mutation, and hence the combined influences of selection and drift, and thereafter – potentially at least – adaptation and evolutionary change. In essence, the broad aim of the paper is to show that insects are genetically capable of adapting and evolving within an ecological context, and indeed do so, and that such changes may be seemingly rapid (perhaps within one or a very few generations in the case of aphids), aided by their often fast reproduction rates relative to many other animal groups.
So called ‘genetically identical’ clones – setting the mutational scene
To demonstrate that fast mutational processes are inherent in individual insects, as well as populations, a good place to start is with clones. These are often wrongly assumed to be genetically identical, but there is no empirical supporting evidence, save at a few microsatellite loci (12 at most), and certainly not over the entire genome, which may be large – some 530 Mb (5.3 × 108 nucleotide bases) on four holocentric chromosomes in the pea aphid, Acyrthosiphon pisum (Harris) (http://www.aphidbase.com/aphidbase/community_links/the_pea_aphid). Such a value may be compared with the mutation rate of 10−6–10−9 per gene per generation seen for many coding genes (e.g. Kumar & Subramanian, 2002 estimate an average of 2.2 × 10−9 per gene per year in mammals), and is often very much higher than that for non-coding tandem repeat regions such as mini- and microsatellites (Goldstein & Schlötterer, 1999; see also http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mutations.html). It is thus not surprising that every member of a vertical (= between generations) clone is likely to show some mutational change compared with the parent population, in this case beginning with a single individual or stem mother.
Indeed, in aphids, which mostly breed during the spring and summer by apomictic (= mitotic) parthenogenesis (Blackman, 1980), this is what has been found. Using grape root stock Phylloxera, Daktulosphaira vitifoliae Fitch, Vorwerk and Forneck (2007) examined eight asexual lineages for 15 generations using AFLP markers. They tested 141 loci of which up to 15 were polymorphic. Mutations were found in every generation, even in early ones. Sequencing of 37 selected polymorphic bands confirmed that such mutations were mostly of non-coding origin. Interestingly, five mutated loci were transmitted to the 15th generation. Such variations, especially if they occur in coding regions, are considered to be potentially adaptive (Forneck et al., 2001; see also Loxdale & Lushai, 2003; Lushai et al., 2003).
Genetic processes leading to speciation
Asexuality in aphids and stick insects.
In aphids, it seems that asexuality can evolve from sexual progenitors by three mechanisms: (1) recurrent mutation at the gene(s) controlling sexuality; (2) hybridisation; (3) inheritance of asexuality genes (Delmotte et al., 2001). This has been described in detail by Delmotte et al. (2001), later by Delmotte et al. (2003), where a hybrid origin was claimed for certain asexual mitochondrial DNA lineages of the bird cherry-oat aphid, Rhopalosiphum padi (L.). Rhopalosiphum padi bearing mitochondrial (mt)DNA haplotype I are of this type; instead of winged migrants returning to the primary woody overwintering host bird cherry (Prunus padus) in the autumn under conditions of reduced light and ambient temperature, the genotype apparently remains on Poaceae (grasses and cereals) all year around (Simon et al., 1996; Martínez-Torres et al., 1997). Whatever their exact evolutionary derivation, such obligate asexuals can no longer breed with the parental stock from which they derived (besides the possibility of some sexual ‘leakage’, Delmotte et al., 2001; Simon et al., 2002) and in effect, are a new evolved entity, not quite a new species as such, but nor the old species either.
Similarly, such derivation of asexuals from sexual taxa has recently been described in stick insects of the genus Timema (Phasmatoptera), a genus inhabiting the dry chaparral vegetation of California and feeding on a range of host plant species (see Law & Crespi, 2002; Schwander & Crespi, 2009 for details). Accordingly, the genus has five obligate parthenogens, each parthenogenetic species having ‘a close morphological counterpart which feeds on the same or overlapping host plants but does not overlap with it in range’. (Law & Crespi, 2002). From microsatellite and karyotypic studies and using a previously published mitochondrial phylogeny (Law & Crespi, 2002), the asexual species were considered to have most likely evolved from the sexually reproducing species, one of which was probably of hybrid origin (Schwander & Crespi, 2009). Because of the complete maintenance of heterozygosity between generations, the five asexual species are thought to have derived – like aphids – via apomictic reproduction, possibly abruptly, whereas eggs produced by virgin females of the sexual species are produced via automixis (where, in contrast to apomixis, meiosis is retained involving restoration of the diploid state by duplication or fusion of the female parentally produced gametes; cf. Simon et al., 2003a). The transition from sexuality to asexuality has resulted in both a high allelic diversity as well a high efficiency for parthenogenesis. It is believed that these traits have positively influenced the success of the asexual lineages in competition with their sexual progenitors, more especially in marginal habitats. As for mechanisms of divergence, it is possible that the asexuals, assuming they are apomictic, arose spontaneously following Wolbachia or Cardinium infections, although it is nevertheless still possible – though less likely – that they derived via automictic processes or suppressed recombination (Schwander & Crespi, 2009).
Chromosomal and other genetic changes in aphids.
In aphids, many species show chromosome polymorphisms in terms of number, sometimes extensive (Blackman & Eastop, 2007). For example, in aphids of the genus Trama (predominantly asexual root feeders; Blackman & Eastop, 2000), T. troglodytes (von Heyden) shows 2n = 14–23, T. caudata (dei Guercio) 9–12 and T. maritima (Eastop) 10–14, with some colonies having individuals bearing different karyotypes. Furthermore, this variation was paralleled by differences in the number and distribution of ribosomal DNA (rDNA) arrays revealed by in situ hybridisation (Blackman et al., 2000). Such high intraspecific karyotype diversity contrasts with very low genetic diversity in the same populations as tested using molecular genetic markers (nDNA EF-1α and mtDNA COI and II), arguing for a rapid evolution of karyotype (Normark, 1999). In the case of T. troglodytes, although it feeds on many species of composite plants, no evidence of karyotype-associated host race formation was apparent (Blackman et al., 2000). This apparent lack of host plant specificity in relation to karyotype may be compared with the situation found in the corn-leaf aphid, Rhopalosiphum maidis (Fitch), another predominantly asexual species, which shows clear divergence in relation to host. Thus karyotypic forms 2n = 10 are specific to barley, Hordeum vulgare, whilst those of 2n = (usually ) 8 prefer Sorghum and maize, Zea mays (Brown & Blackman, 1988). It is likely that even in the event of rare sexual crossing of such chromosome forms, hybrids would not be produced due to chromosome non-disjunctions on the metaphase plate during meiosis (Blackman, 1980). Such chromosomal divergence is also seen in species of the genus Amphorophora feeding on raspberry (Rubus idaeus) where 2n = 18 in A. idaei (Börner) and on blackberry (Rubus fruticosus agg.) where 2n = 20 in A. rubi (Kaltenbach). The two species are distinguishable morphologically using multivariate methods (Blackman et al., 1977).
Other cases of evolution of closely related forms are known where the chromosomal number remains the same, but clear levels of sub-specific divergence are seen to be occurring. An example concerns the biotypes of the Greenbug, Schizaphis graminum (Rondani) in North America which show host preference, apparently evolved before the introduction of modern agricultural practices about 5000 years BP, and discriminated using mtDNA cytochrome oxidase subunit I (COI) markers (Anstead et al., 2002). Another perhaps more recent example of divergence concerns aphids of the genus Sitobion feeding on cereals and wild grasses. Studies using polymorphic microsatellite markers have revealed that Sitobion avenae sensu lato (s.l.) populations have different frequencies of alleles on wheat (Triticum aestivum) and cocksfoot grass (Dactylis glomerata) and may be split into three groupings: ‘wheat specific’ multilocus genotypes (= S. avenae s.s.), cocksfoot multilocus genotype lineages and apparently introgressed with the blackberry grain aphid, Sitobion fragariae (Walker) s.s. and interrelated multilocus genotypes found on both hosts. The genotype group with S. avenae and S. fragariae-like alleles found on cocksfoot also carried S. fragariae-like mtDNA in approximately 80% of cases tested, thereby indicating ‘asymmetrical gender hybridisation’ with the holocyclic S. fragariae (Sunnucks et al., 1997). This means that in most cases, males of S. avenae were attracted to sexual females (oviparae) of S. fragariae on cocksfoot, rather than the other way around, probably because the latter species predominates on this host (Loxdale & Brookes, 1990). The sex pheromones of both species are very similar in terms of the two known constituents and their proportions (Goldansaz, 2003).
It is also known that introgression occurs between different species of Myzus aphid as shown from the rDNA internal transcribed spacer (ITS) region: thus some individual peach-potato aphids, Myzus persicae (Sulzer) also bear the ITS region of a closely related species, the black cherry aphid, M. certus (Walker) (Fenton et al., 1998). Furthermore, M. persicae comprises a complex of closely related forms, some differing in chromosome number, morphology, and host preference. For example, whilst M. persicae s.s. (2n = 12) is seemingly polyphagous, attacking plants in 40 families (Blackman & Eastop, 2000), M. antirrhinii (Macchiati) (2n = 13) is specific to snapdragon (Antirrhinum spp.) (Hales et al., 2000). The possibility exists that there are other morphologically similar, host-adapted forms of M. persicae yet to be discovered, in addition to the snapdragon aphid and a form specific to tobacco (Nicotiana tabacum) as described below. Lastly, in relation to M. persicae, insecticide resistance is associated in highly resistant (R2 and R3) forms of the aphid with an autosomal 1,3 translocation (Blackman, 1980; Blackman et al., 1995). In effect, it is a reproductive isolating mechanism separating normal karyotypic forms from highly resistant forms which are obligate asexual. The highly resistant forms have a duplication of the esterase-4 gene (up to 80 fold in R3s; Field et al., 1999) and even without the presence of the translocation, it is difficult to see how genotypes bearing the large amplification (amplicons) could successfully crossover with individuals bearing lower copy numbers.
Additional factors in aphid evolution.
Pea aphids, Acyrthosiphon pisum, like Greenbugs, show host adapted biotypes, here on red clover and alfalfa, which can be discriminated to some extent using allozymes markers, although the distribution of alleles between populations is not absolute, suggesting some degree of gene flow (leakage) between the evolved forms (Via, 1999; Via et al., 2000). This appears to be a case of sympatric speciation in action (Via, 2001), since hybrids of the two forms have a reduced average fitness on reciprocal transfer experiments than individuals on their preferred host plants (Via et al., 2000). Mapping studies by Hawthorne and Via (2001) of host adapted biotypes showed that there are quantitative trait loci (QTL) along the genome which have effects on performance which in turn effect assortative mating through habitat choice, thereby reinforcing genetic segregation on the different (host) habitats. In a recent paper (Via & West, 2008), it was further shown that during the parthenogenetic phase of these aphids, extensive ‘divergence hitchhiking’ occurs around key QTL loci because ‘reduced inter-race mating and negative selection decrease the opportunity for recombination between chromosomes bearing different locally adapted QTL alleles'. This is likely to reinforce evolution by sympatric means. It is possible though, indeed probable, that maladapted genes are carried via hitchhiking with genes under selection, which could retard differentiation (see also Peccoud & Simon, 2010, this issue).
Besides such QTL loci and their importance in the sympatric speciation process, attraction to specific sex pheromones is also apparently an important factor. As mentioned above, M. persicae is a complex of species, some like the snapdragon aphid, M. antirrhinni, differentiable by chromosome number and colour (dark green compared with normal pale green M. persicae s.s); however, other forms also exist whose taxonomy has remained debatable for some time. One such form is the nicotine-feeding form Myzus nicotianae Blackman, probably a true species in its own right (Blackman, 1987). Myzus nicotianae is morphologically similar to M. persicae s.s. (Margaritopoulos et al., 2000) and is usually isolated from it because either or both taxa reproduce predominantly parthenogenetically in many parts of the world (Margaritopoulos et al., 2007). However in Greece, where peach trees, Prunus persicae, abound, both species go through a sexual phase, returning to this host in the autumn from secondary herbaceous spring and summer hosts, where the sexuals (winged males and wingless oviparae) mate, whereupon the latter produce cold hardy overwintering eggs (Blackman, 1980). Use of microsatellites has shown the greatest genetic divergence to exist between populations in tobacco and non-tobacco growing regions, irrespective of reproductive mode. Following statistical analysis, populations could be split into three groups: persicae, bisexual nicotianae, and unisexual nicotianae. Calculation of genetic distances between populations revealed regional differentiation with marked temporal (between year) stability, suggesting a low level of inter-population gene flow (= migration ). Very interestingly, it was also found that assortative mating between the taxa was promoted by differences in the daily rhythm of female signalling, peak activity coinciding with periods of sub-specific male searching, males showing greater attraction to the sex pheromone of their own, rather than the alternative subspecies. Thus, these additional mechanisms enhance further genomic divergence, reinforcing speciation (Margaritopoulos et al., 2007). In the host adapted races of pea aphids, some of the sexuals of which are both wingless, males are differentially attracted to their sub-specific females (via sex pheromones), hence reinforcing evolutionary divergence (Knäbe, 1999). The fact that the different races also have specific endosymbionts is also likely to lead to ecological specialisation (Simon et al., 2003b; Peccoud & Simon, 2010, this issue).
Polyploidy changes and gene amplification changes.
Polyploidy is a common method of population divergence in many taxa, although it seems generally rare in insects and always associated with a parthenogenetic mode of reproduction (Lokki & Saura, 1979). However, weevils (Coleoptera: Curculionidae) are especially adept at using this mechanism of evolutionary change. Such changes have for many years been examined by the group at the University of Helsinki, Finland, originally compromising Suomalainen, Saura, Lokki, and co-workers. Recently, the topic has been revisited by Stenberg et al. (2003) following an extensive molecular study of the black vine weevil, Otiorhynchus scaber L. In this species, three forms have been traditionally recognised: diploid sexuals, triploid, and tetraploid parthenogens. In Europe, all forms can coexist in a small area in the Alps (fig. 1 in Stenberg et al., 2003), but like many asexuals, only the polyploid parthenogens have succeeded in colonising marginal areas. Following phylogenetic analysis based on three partial mitochondrial genes, parthenogenesis and polyploidy were found to have originated at least three times from different diploid lineages: two major mitochondrial lineages were reported, with > 2.5% sequence divergence between the most basal groups, whilst the current distribution and phylogenetic relationships were found to be only weakly correlated. Furthermore, in contrast to previous findings, morphologically indistinguishable diploid clones were found to co-exist with diploid sexual females, and have most probably derived from them. The authors conclude that ‘it is mainly an increase in ploidy level and not the benefits of asexual reproduction that confers to polyploid parthenogens the advantage over their diploid sexual relatives' (Stenberg et al., 2003). As discussed by Lokki and Saura (1979), there are: (1) apparent environmental correlations in the distribution of polyploid forms; (2) most polyploid insects have life cycles extending over two or more years; and (3), they are in general flightless. Probably the consequences of mutations in polyploid lineages, whereby overall heterozygosity is increased and accumulates, gives such forms a competitive edge over sexually reproducing conspecifics, especially in ecologically unstable marginal habitats. However, this advantage may only be temporary in an evolutionary sense, since asexual lineages are also likely to accumulate deleterious alleles by Muller's ratchet, leading to the eventual extinction of given lineages by so-called mutational meltdown, although such extinctions have not been proven in asexual insects (cf. Lushai et al., 2003; Loxdale & Lushai, 2007).
Hybridisation is also a common evolutionary mechanism in both animals and plants, but often not that important since the products are usually effectively sterile. The topic has recently been discussed at length by Mallet (2007). In insects, hybridisation has been especially well studied in stick insects of the Bacillus species group (Phasmida; e.g. see Bullini & Nascetti, 1989). According to Scali et al. (2003), some sexually reproducing species [i.e. Bacillus rossius (Rossi) and B. grandii Nascetti & Bullini] are sharply differentiated in terms of allozyme alleles and hence appear to be largely reproductively isolated. B. atticus Brunner is a polyclonal automictic parthenogen, sister to B. grandii grandii, hybridising to produce diploid (B. whitei = rossius/grandii) or triploid (B. lynceorum = rossius grandii/atticus) clonal forms which reproduce apomictically. It appears that such asexual forms are spontaneously produced either by apomixis, automixis, hybridogenesis (in which the sperm fertilises the egg, but its chromosomes are not transmitted to the next generation; cf. Simon et al., 2003a), or androgenesis (in which diploid offspring carry nuclear chromosomes from the male parent only; cf. McKone & Halpern, 2003), but even the apomictic forms never completely lose the ability for recombination and a modified meiotic programme allows a ‘low but effective’ recombination to occur (see Scali et al., 2003 for details). The authors define those animals that reproduce by parthenogenesis, hybridogenesis, etc., but still make use of egg and meiotic mechanisms, as meta-sexual (Scali et al., 2003).
Butterflies and moths are also known to hybridise. For example, the European budmoth, Zeiraphera diniana Guenée (Tortricidae), has two host preferring forms, larch and spruce. AFLP mapping analysis confirmed the existence of hybridisation along with strong heterogeneity between chromosomes in terms of host race molecular divergence (average FST = 0.216). In contrast, geographically separated populations of the same host race failed to show such heterogeneity. It appears that selection acts at a few linkage groups along the genome, allowing sympatric divergence, whilst homogenising others. Hence such selection driven genetic divergence in the presence of continuing gene flow is a likely feature of a sympatric mode of speciation (Emelianov et al., 2004).
As a good example of mitochondrial introgressive and nuclear hybridisation in butterflies, blues of the genus Lycaeides (Lycaenidae) of Sierra Nevada in the USA may be cited. It appears that a high altitude or alpine Lycaedes is a spontaneous and fertile hybrid of two parent species, Lycaeides melissa (W. H. Edwards) and L. idas (L.). Molecular genetic analysis using mtDNA COI and II and AFLP markers showed that alpine Lycaeides populations have a mosaic genome that comes from both L. melissa and L. idas, its genome being both distinct from, and younger than, those of its parent species, i.e. 0.44 versus 1.9 and 1.26 Myr in L. melissa and L. idas, respectively (Gompert et al., 2006, 2008). In addition, in one particular mitochondrial haplotype (h01), reduced genetic diversity was found relative to nuclear diversity, suggesting that the spread of this genotype was facilitated by selection. It appears that the success of this haplotype relative to other haplotypes may be due to Wolbachia infection: linkage disequilibrium between mitochondrial haplotype 1 and Wolbachia infection status may have resulted in indirect selection favouring the spread of this particular haplotype in the populations studied. The authors conclude by emphasising the importance of introgressive mito-nuclear discordance in revealing that both mitochondrial and nuclear genes have different histories relating to non-neutral selective processes and inheritance (Gompert et al., 2008).
Hosts shifts as mating barriers.
Hosts shifts have already been alluded to above and are well known means by which insects can find new resources and by so doing, decrease inter- and intraspecific competition, including interclonal competition. It is also a means of moving to enemy-free space, thereby avoiding predators, parasites and pathogens (Jeffries & Lawton, 1984). The ‘classic’ example of an insect species shifting hosts in recent historical times is the tethritid fruit fly, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae) of North America which switched from hawthorn to apple around 150 years ago. Partial isolation is maintained by assortative mating and other pre- and post zygotic barriers, including sex pheromones, host plant (including fruit) cues, and allochronic isolation. Bush, Berlocher, Feder, and co-workers have long studied the ecology and genetics of this insect and its evolutionary divergence, along with that of its close relatives (e.g. cf. Feder et al., 1998 for a review; Linn et al., 2003, 2004). Some of the most recent studies examine the co-evolution of the primary hymenopterous wasp parasitoids (Diachasma spp., Braconidae) attacking flies of the Rhagoletis sibling species complex (Feder & Forbes, 2010, this issue).
A second example concerns how climate warming has led to increased genetic introgression across a narrow hybrid zone separating the eastern and Canadian tiger swallowtails in North America, Papilio glaucus L. and P. canadensis Rothschild & Jordan (Mercader et al., 2009). As a consequence, an allochronically separated hybrid population with a delayed emerging phenotype or ‘late’ flight has arisen. In addition, recombination of the parental genomes has seemingly caused a shift in host use from the preferred hosts of P. glaucus–Tulip tree, Liriodendron tulipifera L. and the Common Hoptree, Ptelea trifoliata L.–to a secondary preferred host of both species, American ash, Fraxinus americana L. However, larval host-use abilities as tested in feeding trials revealed a mixture of the two parental species; hence no divergence had actually occurred in terms of larval host use. As the authors state, this ‘scenario represents an instance where a shift in a major ecological trait, host use, is likely occurring as a by-product of a shift in an unrelated trait (delayed emergence) leading to partial reproductive isolation’. (Mercader et al., 2009). As such then, the hybrid is both parapatrically as well as allochronically separated from its parental species, a separation reinforced by the enforced host shift and delayed adult emergence. It thus appears that as in the case of the alpine Lycaeides, a totally new hybrid species has arisen and is flourishing in its new ecological circumstances.
Enzyme-based adaptive changes.
Besides chromosomal and other changes already alluded to, metabolic differences due to the quantitative expression of existing or novel enzymes, especially detoxifying ones, may allow shifts onto novel hosts which may ultimately become adaptive to the point that a new sub-specific forms or ultimately full species arise. Presumably, such a change could occur within one generation in response to a favourable mutation and thus is another means of rapid evolution in insects. An example concerns the substrate-specific cytochrome P450 monooxygenase enzymes CYP6B1 and CYP6B3 in Papilio polyxenes F., which allow larval specialisation on furanocoumarin-containing host plants. In contrast, CYP6B4 and CYP6B17 enzymes in polyphagous butterflies Papilio glaucus and P. canadensis, and which are induced to different extents (Li et al., 2002), have a broader substrate range. Another closely related butterfly, P. multicaudatus (W. F. Kirby), which is oligophagous with one furanocoumarin-containing host, is putatively ancestral to the two polyphagous species. Molecular analysis of these enzymes in P. multicaudatus suggests that loss of specialisation arose from relatively few mutational changes, which in turn has allowed broader catalytic activities, including its ancestral furanocoumarin-metabolising activities (Mao et al., 2007). In another study (Li et al., 2002), 13 CYP6B genes were isolated from both P. glaucus and P. canadensis. These genes appear to be in an early stage of divergence and, as a result of geographical isolation of the two species and differential exposure to chemically distinct host plants, the function of the P450 enzyme genes has been affected, not only in terms of the coding regions, but also their promoter regions.
Pheromone-based changes causing sympatric divergence.
Qualitative and quantitative differences in sex pheromones (pre-mating signals) can cause assortative mating of individuals within natural populations leading to ecological divergence. In the European corn borer moth, Ostrinia nubilalis (Hübner) (Pyralidae) in North America, a major pest of maize, males of two races respond differentially to different doses/ratios of (E)- and (Z)-11-tetradecenyl acetate (E- and Z-11-14:OAc): males of the univoltine Z race display greater specificity and sensitivity to a 3:97 ratio of E to Z acetates, whilst males of the bivoltine E race preferentially respond to a 99:1 E:Z pheromone mix. (Cosséet al., 1995; Linn et al., 1997). The moth was first introduced into Massachusetts in 1917 but may have arrived earlier and has now spread north to Canada and westwards to the Rocky Mountains. Recent mtDNA (COI and II) analysis (Coates et al., 2004) has revealed that significant genetic differences occur between Atlantic coast and mid-western US populations as well as between uni- and bivoltine ecotypes, possibly due to mating period asynchrony and a low level of mating emphasising that the latter entities are definitely evolutionary divergent (see Coates et al., 2004). Such divergence possibly arose as a consequence of allopatric separation at some time in the historically recent past or to a mutation in the moth's olfactory receptors such that males of the two forms responded to different doses/ratios of the female sex pheromone and hence became divergent physically (i.e. reproductively largely isolated), and later, ecologically too [including in terms of other life-cycle traits such as development, survival, and diapause characteristics (Brindley et al., 1975)]. It is also known that racially distinct specific reductase enzyme systems are responsible for the differential production (blend) of the E- and Z- forms of the sex pheromone in this moth (Glover et al., 1987; Löfstedt, 1993; Zhu et al., 1996), and are controlled by a single autosomal gene (female production locus; Klun & Maini, 1979; Dopman et al., 2005), thereby reinforcing the status of the races. However, this gene is apparently not directly linked to the genetic region that controls the male phenomenal behavioural response (Dopman et al., 2004, 2005). Two other loci are responsible for the pheromone polymorphism in terms of male behavioural response and control of the organisation of the male pheromone sensitive sensilla, respectively (Hansson et al. 1987; Glover et al., 1989, 1991; Cosséet al., 1995). According to Cosséet al. (1995), males from the two O. nubilalis pheromone races differ in signals transmitted by olfactory receptor neurons (ORNs) after stimulation with each of the two pheromone components. However, these differences in ORNs' responses to the pheromones map to a completely different genomic location than that governing the male's actual behavioural response.
In other closely related species of moth like the American noctuids Heliothis virescens (F.) and H. subflexa (Guenée), attraction of males to female sex pheromones has also been recently found to be based on qualitative and quantitative differences in sex pheromones doses/ratios, a scenario which maintains specific identity in the face of potential interspecific mating and gene flow in these species (Choi et al., 2005; Groot et al., 2009a; Gould et al., 2010). Even so, during recent chemical ecological studies of natural populations of these moths, significant intraspecific variation was found in the of sex pheromones blend components and ratios of these two moth species, as well as in the male behavioural response, both between geographic regions (sites in North Carolina, Stoneville, Mississippi, Texas, and Mexico) and consecutive years within a region (cf. Groot et al., 2009a for details). The authors conclude that besides purely selective or stochastic events (i.e. genetic drift and migration), such genetic variation seems to be partially controlled by genetic factors whilst being correlated with abiotic factors (i.e. temperature, relative humidity and day length) as well as the presence and abundance of other moth species which ‘may cause communication interactions and thus constitute the semiochemical environment'. The observed temporal variation within a population apparently involves within-generation optimisation of the pheromonal signal governed by physiological adjustments of moths responding to their experience of the local chemical environment.
Complementary work by Groot et al. (2009b) involving QTL analysis linked to the biosynthetic pathways of both H. virescens and H. subflexa has implicated several candidate genes associated with such divergent pheromone pre-mating signals, the most important of which code for the enzymes acetyl transferase, a desaturase(s), and a fatty acyl reductase or alcohol oxidase.
Contact pheromones (CHCs).
As discovered in Drosophila, the enzyme desaturase-2 is involved in the production of long chain fatty acid hydrocarbons, which form a major component of the insect cuticle, and are important in relation to desiccation resistance and species recognition (Ritchie & Noor, 2004; Foley et al., 2007, and references therein). As detailed by these researchers, such cuticular hydrocarbons (termed CHCs) are important for a range of functions in insects and have been studied concerning their roles in mate and species recognition, more especially sexual signalling and recognition, and in Drosophila melanogaster Meigen, in relation to their biosynthesis, genetic regulation, and sex specificity. In the D. melanogaster species complex, they have been shown to be important in sex recognition and sexual isolation between closely related species and races. In this and in many other Drosophila species, mate choice comprises several distinct elements: a courtship dance, wing song, and ‘assessment of the CHCs of a potential mate through olfaction, gustation, and chemoreceptors in the front legs' (Foley et al., 2007; reviewed earlier by Greenspan & Ferveur, 2000). These compounds are also thought to be associated with assortative mating, both at the strain, race, and among species level. Thus CHCs are seen to be a powerful means by which insect species (here Drosophila) can recognise their mates, court them appropriately and mate with them assortatively, reinforcing and thereby maintaining ecological strain-race differences and higher levels of evolutionary divergence and fidelity.
Wolbachia, a group of intracellular inherited bacteria that infect a wide range of arthropods, are well known to be associated with a variety of reproductive alterations in their hosts, more especially cytoplasmic incompatibility, usually involving sterility or feminisation of males and in some cases, parthenogenesis as a direct result of elimination of the male sex (Zimmer, 2001; Tortora et al., 2007). It is also thought that the bacteria can be passed between individuals of the same generation by horizontal transmission due to rare hybridisation events or by transmission via hymenopterous wasp parasitoids (Cook & Butcher, 1999; but see West et al., 1998). In mosquitoes, the Wolbachia pipientis assemblage is divided into two major groups (A and B) and 12 subgroups. The bacteria were detected using PCR assays involving specific primers in ∼30% of wild-caught mosquito species except Anopheles: group B Wolbachia strains showed more phylogenetic concordance with their host taxa than A strains (Kittayapong et al., 2000). It is possible that such infections, by preventing random mating among specific taxa, lead to divergence, certainly in terms of the production of closely related asexual taxa, and perhaps in sexual taxa where the normal sex ratio is disrupted, thus enhancing the evolution of female host preferring strains or races. One can imagine this happening in Lepidoptera, for example, where the females are the heterogametic sex. Certainly, in members of the African Queen butterfly, Danaus (Anosia) chrysippus (L.) (s.l.) species complex, mtDNA COI and nDNA data in sympatric and parapatric subspecies revealed extensive hybridisation to take place, thereby producing what are effectively new nascent species (Lushai et al., 2005). The authors show that ‘hybridism among lineages in sympatry is currently enforced, in the face of assortative mate choice, by a bacterial symbiont, Spiroplasma, a male-killer that forces females in female-biased populations to pair with heterotypic males'. From this, they claim that ‘neither D. (A.) chrysippus s.l. as presently circumscribed, nor its component clades, conform to any established concept of species'. This topic is discussed by Smith et al. (2010, this issue).
Phenotypic plasticity as a source of genetically-based adaptive changes.
In insects showing phenotypically plastic responses, the gene/s concerned are (apparently) the same, but the expression involves variation of the phenotype which may have adaptive, and hence, evolutionary significance. The topic is explored at length by Whitman and Agrawal (2009) and, along with describing how phenotypic plasticity contributes to the speciation process, includes a section on how stress induces adaptive mutations in individuals. Such stressors include transposable elements or lowered immunity, which may allow mutation-inducing viruses to affect the genome. Some of the plastic variations mentioned seem almost Lamarckian in their effect, and follow the doctrine of Baldwin (1896), namely that ‘The most plastic individuals will be preserved to do the advantageous things for which their variations show them to be the most fit, and the next generation will show an emphasis of just this direction in its variations'. However, ultimately, natural selection selects for the most beneficial plastic responses in terms of their contribution to fitness (Whitman & Agrawal, 2009). Yet, such selection can lead presumably to the evolution of very different forms in the same species (e.g. the population density-induced phase changes in locusts) to actual incipient speciation. Once sympatric divergence has reached the latter level, then perhaps full speciation is inevitable, as behavioural mate recognition–assortative mating factors (and involving CHCs and perhaps specific sex pheromones) act and make the evolutionary return journey unlikely, or impossible. In this sense, such populations have crossed their ecological ‘Rubicon’, never to return, for better or worse.
Epigenetic changes and transgenerational effects.
Whitman and Agrawal (2009) discuss this topic in relation to the offspring phenotype produced as a result of transgenerational phenotypic plastic effects. These effects possibly include repeated cycles of habitat induction or imprinting leading to habitat-specific adaptation (Davis & Stamps, 2004) and mutualistic endosymbiotic bacteria, such as Buchnera in aphids. Peccoud and Simon (2010, this issue) discuss habitat-specific adaptation in relation to the ecological specialisation of pea aphids, Acyrthosiphon pisum (s.l.), the first step on the road to evolutionary divergence leading to full speciation. Lastly, the transgenerational induction of wings in aphids as a result of stress (overcrowding or via alarm pheromone mediation leading to so-called ‘pseudo-crowding’ effects), may arise due to epigenetic effect, e.g. DNA methylation. This phenomenon is discussed by Kunert et al. (2010, this issue). That the stressors may be acting directly upon the two generations of embryos within the adult aphid (i.e. telescoping of generations; cf. Dixon, 1998), means that the effects is not truly transgenerational, but rather inductive. Even so, it is nevertheless a subsequent generation in which the genetic–phenotypic change is induced, and this new form is henceforth subject to the vagaries of selection, and in turn, adaptation and evolution. Maybe the phenomenon is not exactly ‘the inheritance of acquired characters' per se, but more near-Lamarckian in its influence, a subtlety that Darwin (and no doubt Wallace) would probably have appreciated and accepted.
As mentioned in the Introduction, that insects represent such an enormous pool of global biodiversity makes them excellent models for the study of evolution, both theoretically and empirically. Of the various individual/population divergence mechanisms discussed, chromosomal changes and host shifts may be considered the most important. The ecology and evolution of host shifts have been demonstrated in a large number of studies of a variety of insects, but especially, as presently described, the fruitfly Rhagoletis, the pea aphid, A. pisum, and in maize aphids, R. maidis, all apparently sympatrically. Not discussed are the numerous chromosomal studies of a number of insect species groups in relation to population divergence, notably including Drosophila, e.g. in relation to inversion polymorphisms (caused by transposable element ‘hotspots’) as a mechanism of reproductive isolation and hence ecological/evolutionary divergence (Cáceres et al., 1999; Ortiz-Barrientos et al., 2002; Noor et al., 2007). Furthermore, as also earlier alluded to, interest in other mechanisms is increasing, especially in pheromone studies, as with the moths O. nubilalis and Heliothis spp., and in CHCs, mainly in Drosophila so far.
Whatever the exact mechanism of population isolation and divergence, and of course, in insects this is often enhanced by their fast rates of reproduction compared with other taxa (e.g. in aphids, a generation can be a week to 10 days!), unless the forms produced by any given mechanism are fertile and able to meet like-mutated individuals, then the process – be it allopatric, parapatric or sympatric/allochronic – is likely not to succeed (White, 1978; Coyne & Orr, 2004). But of course, this is true of all mutants in an established population giving rise to new ecological–evolutionary entities. It is something that students of inbreeding seem often to forget, i.e. that established populations with perhaps extremely high effective population sizes may give rise to new populations by foundation events, bottlenecks, etc., with extremely low effective population sizes (Loxdale, 2002, 2007a,b, 2008). Yet this is the process we believe has gone on since the Earth became a suitable habitat for life in all its richness and extravagant diversity, and initiated the interactions that led to ecological processes and specialisations, eventually evolution, including of insects.
I especially thank Professor Mike Claridge and Drs Astrid Groot and Jane Hill for their very helpful comments on earlier versions of the manuscript.
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